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Title: An investigation into the removal of human topoisomerase II-DNA adducts
Author: Curtis, Hannah
ISNI:       0000 0004 2719 9267
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2008
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Human topoisomerase 11 (topo 11) is an essential enzyme that controls DNA topology by relieving positive and negative supercoiling. It does this by creating a transient double-stranded break in one DNA duplex to allow a second duplex to pass through. The DNA double-stranded break is bridged by the topo 11 enzyme which is attached to each DNA end via a 5' covalent phosphotyrosyl bond. Human cells express two distinct topo JI isoforms, topo JI a and topo JI ~. The anti-cancer drug etoposide is extensively used in the treatment of malignancies. Etoposide traps the topo II homodimer in its normally transient cleavage state, a permanent DNA double stranded break can be produce through cellular processing. Non-homologous end joining (NHEJ) is a major pathway for the repair of to po II- mediated DNA damage, and inhibition of DNA-PK potentiates the cytotoxicity of topo JI poisons. The topo II enzyme remains covalently attached to the broken DNA and must be removed for NHEJ to take place. No mechanism to date has been elucidated for the removal of covalently attached human topo II-l)NA adducts. Inhibition of this process would slow the repair of to po II damage, thus potentiate the effects of to po IJ-targeting drugs. The Irapped in AgaRose DNA [mmunojitaining (TARDIS) assay was successfully adapted to incorporate a protein incubation phase to test candidate proteins for their ability to remove etoposide-stabilised human topo U-DNA adducts. Mrell and Tdp 1 immunoprecipitates from human chronic myelogenous leukaemia cells were identified that were capable of removing topo 11 complexes. Proteomic analysis identified nucleases and DNA repair proteins that were present in the two immunoprecipitates. Results provide evidence for the first time that Mre I I can facilitate the removal of human topo IJ a DNA adducts. In addition, results suggest that human topo II-DNA adducts are also removed by a 5' phosphodiesterase. These results may provide exciting new targets for future leukaemia drugs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available